Fluorescent probes for imaging biological phenomena have been actively developed in researches of life chemistry in recent years, and various fluorescent probes for measurement of pH, various metal ions, active oxygen species, and the like have been proposed. For example, as fluorescent probes for measuring pH, there have been proposed BCECF (2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein) and derivatives thereof, CFDA (carboxyfluorescein diacetate) and derivatives thereof, SNARF-1(seminaphthorhodafluor) and derivatives thereof [the catalogue of Molecular Probe (Handbook of Fluorescent Probes and Research Chemicals, Tenth Edition), Chapter 20(pH indicators), for all the compounds]. Further, as fluorescent probes for measuring zinc ion, there have been proposed TSQ (Reyes, J.G., et al., Biol. Res., 27, 49, 1994), zinquin ethyl ester (Tsuda, M., et al., J. Neurosci., 17, 6678, 1997), dansylaminoethylcyclen (Koike, T., et al., J. Am. Chem. Soc., 118, 12696, 1996), and Newport Green [the catalogue of Molecular Probe (Handbook of Fluorescent Probes and Research Chemicals, Tenth Edition), Chapter 19(Calcium ion, magnesium ion, zinc ion and other metal ions)], and further, there have been proposed, by the inventors of the present invention, a zinc ion probe comprising fluorescein as a fluorescent mother nucleus (Japanese Patent Laid-open Publication (KOKAI) No. 2000-239272, International Patent Publication WO01/062755, Japanese Patent Laid-open Publication No. 2004-315501), an zinc ion probe comprising benzofuran as a fluorescent mother nucleus (International Patent Publication WO02/102795), and the like.
Although fluorescent characteristics of these fluorescent probes desirably change only when they react with a target measurement object, they are often influenced by other factors. In particular, application of fluorescent probes to cells or biological tissues has a problem that it involves many factors which affect the measurement. For example, concentration of a fluorescent probe introduced into cells may vary depending on type of the cells, intensity of excitation light may vary in measurement regions depending on thickness of cell membrane, a fluorescent probe may localize at a highly hydrophobic portion such as membranes, and the like.
As a method for reducing measurement errors induced by these factors to realize accurate quantitative analysis, the ratio method has been developed and used (Kawanishi Y., et al, Angew. Chem. Int. Ed., 39(19), 3438, 2000). This method comprises the step of measuring fluorescence intensities at two different wavelengths in a fluorescence spectrum or an excitation spectrum to detect a ratio thereof. In this method, influence of concentration of the fluorescent probe itself or intensity of excitation light can be ignored, and measurement errors can be eliminated, which may be caused by localization of the fluorescent probe itself, change of concentration thereof, discoloration thereof, or the like, when the measurement is performed at one wavelength. In order to enable the above ratio measurement, there is needed a fluorescent probe which shows change in excitation light wavelength or fluorescence wavelength before and after a reaction with a measurement object. For example, the pH fluorescent probe SNARF-1 has a property that the peak of fluorescence wavelength shifts to the longer wavelength side due to deprotonation when pH shifts to the alkaline side, and when it is excited around 500 nm, fluorescence intensity around 580 nm decreases with increase of pH, whilst fluorescence intensity around 640 nm increases with increase of pH. Therefore, if a solution containing this compound is irradiated with an excitation light, and ratio of fluorescence intensities measured at that time at appropriate two wavelengths is obtained, pH can be accurately measured regardless of probe concentration, intensity of light source, size of cells, and the like. Further, the zinc ion fluorescent probe described in International Patent Publication WO02/102795 is a fluorescent probe based on the principle of intramolecular charge transfer, and uses or shows an excitation wavelength of 354 nm and a fluorescence wavelength of 532 nm when it does not capture zinc ion, but shows about 20 nm blue shift of the peak in the excitation spectrum depending on concentration of zinc ions. Therefore, by using wavelengths of 335 nm and 354 nm as the excitation wavelengths, measuring fluorescence intensities at those excitation wavelengths, and obtaining the ratio of the intensities, zinc ion concentration can be accurately measured irrespective of the probe concentration, light source intensity, size of cells, and the like. However, a problem arises that they are ratio type fluorescent probes utilizing or showing excitation light wavelength/fluorescence wavelength of the visible light region or shorter, and such excitation light shows poor permeability into biological tissues. Furthermore, they fail to solve the problem that measurement is easily influenced by autofluorescence of cells themselves.
Cyanine dyes are widely used in various fields, and they are also used in the field of fluorescence imaging for studying physiological functions as fluorescence labels of biological molecules. Among these cyanine dyes, tricarbocyanine type dyes are fluorescent dyes showing a maximum absorption wavelength and maximum fluorescence wavelength in the near infrared region of around 650 to 950 nm, of which lights are comparatively less absorbed by biological molecules, and thus they have an advantage that they allow use of lights of a wavelength which can penetrate into deep parts of biological tissues. In addition, since biological substances scarcely emit autofluorescence of the near infrared region, the characteristics of tricarbocyanine type dyes are suitable for in vivo imaging.
In addition to cyanine type dyes for directly labeling biological molecules with fluorescence, tricarbocyanine dyes which specifically react with biological molecules to change fluorescence intensity thereof have recently been developed. Examples include a near infrared fluorescent probe for calcium ions (Ozmen, B., et al., Tetrahedron Lett., 41, pp. 9185-9188, 2000), and a near infrared fluorescent probe for nitrogen monoxide (NO) (WO2005/080331). These fluorescent probes are probes showing only fluorescence intensity changes, which do not show changes of excitation wavelength and fluorescence wavelength, before and after a specific reaction with a biological molecule. Moreover, as pH probes, there are the compounds described in International Patent Publication WO00/75237 and CypHer (GE Healthcare Bioscience). They show increase of fluorescence intensity with decrease of pH in the neutral region or lower pH region on the basis of the principle that the nitrogen atom of the nitrogen-containing hetero aromatic ring bonded to the polymethine chain of the cyanine structure is protonated to emit fluorescence. Furthermore, the inventors of the present invention recently developed a near-infrared fluorescent probe for zinc ion (Japanese Patent Application No. 2005-057265) and a near-infrared fluorescent probe used for pH measurement (Japanese Patent Application No. 2007-035768). These are ratio fluorescent probes of which excitation wavelength changes depending on changes of pH or concentration of zinc ion. They show a maximum absorption wavelength of 650 to 800 nm, which wavelength shows superior permeability into biological tissues, and a marked wavelength shift in the peak of excitation spectrum according to change of zinc ion concentration or pH, therefore, by irradiating them with excitation lights of appropriate two wavelengths of 650 to 800 nm, and obtaining ratio of fluorescence intensities measured at the wavelengths, zinc ion concentration and pH in a deep part of biological tissue can be highly accurately measured.
Further, since fluorescence change by fluorescence resonance energy transfer (FRET) may also be change of two wavelength ratio type, the ratio measurement is also possible with a FRET type fluorescent probe, which makes it possible to quantitatively measure a measurement object irrespective of probe concentration, light source intensity, size of cells, and the like. However, most of conventionally known fluorescent probes utilizing FRET (for example, the fluorescent probe described in Japanese Patent Laid-open Publication No. 2000-316598 and the like) utilize fluorescence of the visible region, and almost no fluorescent probes utilizing or showing an excitation light wavelength/fluorescence wavelength of the near infrared region are known, except an FRET type pH probe described in International Patent Publication WO00/075237 which utilizes on/off of fluorescence in connection with pH change. This probe described in International Patent Publication WO00/075237 has a problem that it can be used only as a pH probe, and it cannot be applied to a probe for an substance to be measured such as other metal ions and enzyme substrates.